Most eukaryotic genes encode mRNA that is monocistronic. Recent evidence indicates, however, that there exists a class of eukaryotic genes which encode bifunctional mRNA. Alternate use of multiple inframe initiation codons allows ribosomes scanning these transcripts to translate more than one protein product. The goal of this proposal is to define how bifunctional mRNA are used by the eukaryotic cell to regulate gene expression. Using oligonucleotide-directed in vitro mutagenesis, three series of mutants will be constructed. The first series of mutants will investigate the factors which influence the efficiency of translation initiation at alternate AUGs of bifunctional mRNA in the yeast gene MOD5. Mutations of the 5' untranslated leader sequence and the initiation codon context will define the role of these factors in the translation of MOD5 transcripts. The second mutant series will examine the transcripts of another sorting isozyme, TRM1, to determine if bifunctional mRNA participates in regulating the expression of the TRM1 products. Transcriptional start sites will be altered such that only the long TRM1 transcript which contains multiple initiation codons is transcribed. the products of this transcript will then be isolated from cells transformed with the mutants and analyzed to determine if the transcript is indeed bifunctional. Finally, a model for the translation of bifunctional eukaryotic mRNA will be tested in mutant HIS4 transcripts. The leader lengths and initiation codon contexts of these mutants will be altered such that if the model is correct, scanning ribosomes will translate the monofunctional messages as if they are bifunctional.